Compositions and devices of poly-4-hydroxybutyrate

ABSTRACT

Compositions of P4HB with high purity have been developed. The compositions are prepared by washing P4HB biomass prior to solvent extraction, and precipitating P4HB from solution. The same solvent is preferably used to wash the P4HB biomass, and as a non-solvent to precipitate the polymer from a P4HB solvent solution. The highly pure P4HB compositions are suitable for preparing implants. The implants may be used for the repair of soft and hard tissues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/529,217, filedOct. 31, 2014, which claims the benefit of priority to U.S. Ser. No.61/900,348, filed on Nov. 5, 2013, the disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to medical devices ofpoly-4-hydroxybutyrate, the compositions used to produce these medicaldevices, and the processes used to produce these compositions. Themedical devices can be used in many types of implant applicationsincluding wound management, general surgery, hernia repair, nerverepair, tissue engineering, orthopedic, craniomaxillofacial surgery,drug delivery, cardiovascular, vascular, cardiology, urology,gynecology, dental, imaging, ear, nose and throat surgery, plastic andcosmetic surgery, and oral surgery.

BACKGROUND OF THE INVENTION

Poly-4-hydroxybutyrate (P4HB) and copolymers thereof can be producedusing transgenic fermentation methods, see, for example, U.S. Pat. No.6,548,569 to Williams et al., and are produced commercially, forexample, by Tepha, Inc. (Lexington, Mass.). Poly-4-hydroxybutyrate(P4HB, TephaFLEX® biomaterial) is a strong, pliable thermoplasticpolyester that, despite its biosynthetic route, has a relatively simplestructure as shown below.

The polymer belongs to a larger class of materials calledpolyhydroxyalkanoates (PHAs) that are produced by numerousmicroorganisms (see, for example, Steinbüchel A., et al. Diversity ofBacterial Polyhydroxyalkanoic Acids, FEMS Microbial. Lett. 128:219-228(1995)). In nature these polyesters are produced as storage granulesinside cells, and serve to regulate energy metabolism. They are also ofcommercial interest because of their thermoplastic properties,biodegradability and relative ease of production.

The PHA polymers have been divided into three classes based on thenumber of carbon atoms in their subunits. Short-chain-length PHApolymers (or scl-PHAs) are made from monomers of 3 to 5 carbon atoms.Medium-chain-length PHA polymers (mcl-PHAs) contain 6 to 14 carbons intheir monomeric units, and long-chain-length PHAs (lcl-PHAs) havemonomers with more than 14 carbons. The properties of these polymersvary dramatically depending upon their chain length, including theirsolubilities, thermal, and mechanical properties. P4HB has four carbonatoms in its monomeric unit, and is therefore classified as a scl-PHA.

Chemical synthesis of P4HB has been attempted, but it has beenimpossible to produce the polymer with a sufficiently high molecularweight that is necessary for most applications (see Hori, Y., et al.,Polymer 36:4703-4705 (1995); Houk, K. N., et al., J. Org. Chem., 2008,73 (7), 2674-2678; and Moore, T., et al., Biomaterials 26:3771-3782(2005)). In fact, it has been calculated to be thermodynamicallyimpossible to chemically synthesize a high molecular weight homopolymerunder normal conditions (Moore, T., et al., Biomaterials 26:3771-3782(2005)).

U.S. Pat. Nos. 6,245,537, 6,623,748, 7,244,442, and 8,231,889 describemethods of making PHAs with low levels of endotoxin. U.S. Pat. Nos.6,548,569, 6,838,493, 6,867,247, 7,268,205, 7,179,883, 7,268,205,7,553,923, 7,618,448 and 7,641,825 and WO 2012/064526 describe use ofPHAs to make medical devices. Methods to control molecular weight of PHApolymers have been disclosed by U.S. Pat. No. 5,811,272 to Snell et al.

PHAs with controlled degradation and degradation in vivo of less thanone year are disclosed by U.S. Pat. Nos. 6,548,569, 6,610,764,6,828,357, 6,867,248, and 6,878,758 to Williams et al. and WO 99/32536to Martin et al. Applications of P4HB have been reviewed in Williams, S.F., et al., Polyesters, III, 4:91-127 (2002), and by Martin, D. et al.Medical Applications of Poly-4-hydroxybutyrate: A Strong FlexibleAbsorbable Biomaterial, Biochem. Eng. J. 16:97-105 (2003). Medicaldevices and applications of P4HB have also been disclosed by WO 00/56376to Williams et al. Several patents including U.S. Pat. Nos. 6,555,123,6,585,994, and 7,025,980 describe the use of PHAs in tissue repair andengineering. U.S. Pat. Nos. 8,034,270, 8,016,883, 8,287,909, WO2011/119742 and WO 2011/159784 disclose fibers, non-wovens, and textilesmade by melt extrusion or dry spinning of P4HB.

German Patent No. DE 3937649A1 to Steinbüchel et al. disclosesproduction of P4HB by fermentation. The P4HB polymer was identified bymethanolysis of the biomass using gas chromatograpy. The P4HB polymerwas not however purified from the biomass.

Two patent applications, WO 99/32536 to Martin and WO 00/56376 toWilliams disclose a method to produce P4HB in recombinant Escherichiacoli K12. The resulting biomass containing the P4HB polymer wasfluidized and lyophilized, and the P4HB polymer extracted withtetrahydrofuran, filtered, precipitated, redissolved in solvent,filtered again, precipitated, washed, and lyophilized. After thispurification process, the P4HB polymer was reported to have thefollowing composition by elemental analysis: carbon 55.63%, hydrogen7.41%, oxygen 37.28%, and nitrogen 41 ppm.

P4HB polymer has also been produced by the methods disclosed by EP2534141 A1 to Van Walsem, and by WO 2013/023140 to Van Walsem.

Production of P4HB homopolymer from glucose has been reported by Zhou etal. Hyperproduction of poly(4-hydroxybutyrate) from glucose byrecombinant Escherichia coli, Microb. Cell Fact. 11:54 (2012). The P4HBpolymer was purified from the biomass.

Several other patents have disclosed methods to purify other PHApolymers from biomass, but none of these other PHA compositions ormethods of purification are currently used to produce medical implantscleared or approved by the US Food and Drug Administration (FDA). U.S.Pat. No. 5,110,980 to Ramsay et al. discloses using hypochloritesolution to digest biomass in order to extract poly-3-hydroxyalkanoates.U.S. Pat. No. 5,942,597 to Noda et al. discloses solvent extraction ofPHA polymers with melt temperatures of about 80° C. or higher frombiomass. U.S. Pat. No. 6,043,063 to Kurdikar et al. discloses directsolvent extraction of certain PHA polymers from biomass withnon-halogenated solvents. U.S. Pat. No. 6,087,471 to Kurdikar et al.discloses the use of pressure and high temperatures to solvent extractPHA polymers. U.S. Pat. No. 7,070,966 to Schumann et al. disclosesmethods to reduce the biomass and enzymatically decompose it. U.S. Pat.No. 7,098,298 to Kinoshita et al. discloses extracting PHA polymers witha monohydric alcohol having 4 to 10 carbon atoms. U.S. Pat. No.7,118,897 to Narasimha et al. discloses the extraction of PHA polymerswith solvents at high temperatures and under pressure, including the useof ethanol to extract PHA polymers. U.S. Pat. No. 7,226,765 to Narasimhaet al. discloses the solvent extraction of PHA polymers at hightemperatures. U.S. Pat. No. 7,252,980 to Walsem et al. discloses solventextraction of PHA polymers, and recovery using centrifugation. U.S. Pat.No. 7,393,668 to Yanagita et al. discloses a method to extract PHApolymers from biomass by physical disruption in the presence of alkalifollowed by treatment of the separated PHA polymer with an enzyme and/ora surfactant to remove impurities adhering to the PHA polymer. U.S. Pat.No. 7,435,567 to Osakada et al. discloses methods to purify PHA polymersby digesting nucleic acids with hypochlorous acid. U.S. Pat. No.7,576,173 to Walsem et al. discloses the extraction of PHA polymers withcombinations of solvents. U.S. Pat. No. 8,357,508 to Mantelattodiscloses a method to extract PHA polymers from biomass by injecting PHAsolvents in liquid and vapor form into the biomass and heating.

The use of methanol to prewash Pseudomonas putida and Ralstonia eutropha4-hydroxyvalerate-containing PHA polymer biomass prior to solventextraction has been disclosed by Gorenflo et al. “Development of aprocess for the biotechnological production of4-hydroxyvalerate-containing polyesters and characterization of theirphysical and mechanical properties”, Biomacromolecules 2:45-57 (2001).The use of methanol to prewash Pseudomonas putida KT2440 biomasscontaining medium chain length PHA polymers has also been disclosed byJiang et al. “Acetone extraction of mcl-PHA from Pseudomonas putidaKT2440”, J. Microbiol. Meth. 67:212-219 (2006). However, impurities withUV absorbances at 241 and 275 nm from the mcl-PHA polymer were stillpresent following the use of methanol as a prewash step, and multipleadditional steps were required to reduce their presence. The authors didnot further identify these contaminants, but commented that nucleicacids and aromatic acids are known to absorb at these wavelengths.Moreover, methanol is highly toxic to humans in small quantities. Invivo, methanol is metabolized via formaldehyde to formic acid, which cancause permanent blindness by the destruction of the optic nerve.Ingestion, inhalation, or absorption of methanol can be fatal. For thesereasons, the use of methanol in the preparation of implantable productsshould be avoided, and its use in pharmaceuticals is restricted andregulated by the FDA as a Class 2 solvent (International Conference onHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH) guidance for industry Q3CImpurities: Residual Solvents, 1997).

In the manufacture of implants using polymers, it is desirable that thepolymeric materials have the lowest levels of impurities possible inorder to prevent or minimize the reaction of the body to the impurities.Such undesirable reactions can include inflammation, cytotoxicity,irritation, pyrogenicity, genotoxicity, carcinogenicity, and acute,subchronic and chronic toxicity. Impurities may be placed in three broadcategories, namely, organic impurities, inorganic impurities, andresidual solvents. The purification of PHA polymers to a level wherethey are suitable for use in implants is particularly difficult due totheir production in biological systems. Such production requires thatthe purification process remove a wide range of impurities, including,for example, lipids, proteins, peptides, heavy metals, endotoxin,polysaccharides, nucleic acids, amino acids, cell wall components,residual feed stocks, and residual media components if the PHA polymersare derived by fermentation. The latter can include yeast extract, soypeptone, antifoam agents, antibiotics, salts, amino acids, trace metals,sugars, and buffers. The purification is further complicated by theknown affinity of PHA polymers for proteins, their relatively lowsolubility or lack of solubility in most solvents, and the difficultiesof removing solvents from polymers to acceptable levels. And thesedifficulties must all be overcome while still yielding PHA polymers withgood yields.

In order to improve the purity and biocompatibility of P4HB, it isdesirable to identify new methods of purification that yield P4HB withreduced levels of: lipid, residues on ignition, nitrogen content, heavymetals, and residual solvent.

It is therefore an object of the present invention to providecompositions of P4HB with improved purity.

It is another object of the present invention to provide methods toproduce P4HB with improved purity.

It is a further object of the present invention to provide implants madefrom P4HB compositions with improved purity.

It is still another object of the present invention to provide methodsfor human or animal use of P4HB implants with improved purity.

SUMMARY OF THE INVENTION

Compositions of P4HB with high purity have been developed. Thecompositions contain carbon, hydrogen or oxygen isotopes (e.g., C¹²,C¹³, C¹⁴), oxygen (e.g., O¹⁶, O¹⁸), and/or hydrogen (e.g., deuterium,tritium) in their natural isotopic ratios, or are enriched for one ormore isotopes of carbon, hydrogen or oxygen. The compositions areprepared by washing P4HB biomass prior to solvent extraction, thenprecipitating the P4HB from solution.

Methods have been developed that allow P4HB to be recovered from P4HBbiomass with the following benefits: (i) higher purity, wherein thepolymer contains less than 100 ppm of lipid assayed as palmitate andless than 40 ppm of nitrogen; (ii) a good yield of polymer with arecovery of greater than 75% of the polymer from the biomass; (iii)minimal loss of polymer molecular weight during recovery such that thepolymer does not lose more than 10% of its weight average molecularweight during recovery; (iv) fewer recovery steps; (v) reduced solventusage during extraction; (vi) easier drying of the polymer; (vii) lowercost; and (viii) faster overall process.

The highly pure P4HB compositions are suitable for preparing implants.The implants may be used for the repair of soft and hard tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show standard curve for the GC-MS determination of serum4HB (1A) and serum concentration of 4HB in test and control animals(1B).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Poly-4-hydroxybutyrate” as generally used herein means a homopolymer of4-hydroxybutyrate units. It may be referred to herein as P4HB. The P4HBcan contain carbon, hydrogen and oxygen in their natural isotopicratios, as well as polymers in which one or more isotopes are enriched.

“Copolymers of poly-4-hydroxybutyrate” as generally used herein meansany polymer of 4-hydroxybutyrate with one or more different hydroxy acidunits.

“Bioactive agent” is used herein to refer to therapeutic, prophylactic,and/or diagnostic agents. It includes without limitation physiologicallyor pharmacologically active substances that act locally or systemicallyin the body.

“Biocompatible” as generally used herein means the biological responseto the material or device being appropriate for the device's intendedapplication in vivo. Any metabolites of these materials should also bebiocompatible.

“Blend” as generally used herein means a physical combination ofdifferent polymers or components, as opposed to a copolymer comprised oftwo or more different monomers.

“Carbon content” as used herein refers to the mass percentage ofelemental carbon in a sample, and is determined by combustion analysis.

“Deuterated P4HB” referred to herein as D-P4HB.

“Endotoxin content” as used herein refers to the amount of endotoxinpresent in a sample, and is determined by the limulus amebocyte lysate(LAL) assay.

“Hydrogen content” as used herein refers to the mass percentage ofelemental hydrogen in a sample, and is determined by combustionanalysis.

“Heavy metal content” as used herein refers to the mass percentage ofheavy metals in a sample, and is determined by the method of the UnitedStates Pharmacopeia (USP)<231>.

“Lipid content” as used herein refers to the concentration of lipids inthe sample, and is determined by GC analysis after butanolysis, and isexpressed in part per million (ppm) palmitic acid.

“Molecular weight” as used herein, unless otherwise specified, refers tothe weight average molecular weight (M_(w)), not the number averagemolecular weight (M_(n)), and is measured by GPC relative topolystyrene.

“Nitrogen content” as used herein refers to the mass percentage ofelemental nitrogen in a sample, and is determined by the Kjeldahl methodof nitrogen analysis, and is expressed in parts per million (ppm).

“Residual solvent content” as used herein refers to the amount ofsolvent in a sample, and is determined by headspace GC-MS, and isexpressed in ppm.

“Residue on ignition” as used herein refers to the amount of residualsubstance not volatized from a sample when it is ignited in the presenceof sulfuric acid, and as determined by the method of the United StatesPharmacopeia (USP)<281>.

“Resorbable” as generally used herein means the material is broken downin the body and eventually eliminated from the body. The terms“resorbable”, “degradable”, “erodible”, and “absorbable” are usedsomewhat interchangeably in the literature in the field, with or withoutthe prefix “bio”. Herein, these terms will be used interchangeably todescribe material broken down and gradually absorbed or eliminated bythe body, whether degradation is due mainly to hydrolysis or mediated bymetabolic processes.

“Sulfur content” as used herein refers to the mass percentage ofelemental sulfur in a sample, is measured by inductively coupled plasmaoptical emission spectroscopy and is expressed in ppm.

II. Composition

Provided herein are compositions containing P4HB, recovered from a P4HBbiomass. PHA4400 or TephaFLEX® biomaterial is a homopolymer of4-hydroxybutyrate manufactured by Tepha, Inc., Lexington, Mass.

The compositions can include known isotopes of carbon, hydrogen andoxygen in their natural isotopic ratios, or one or more isotopes whichare enriched. The P4HB compositions have the following benefits: (i)higher purity; (ii) minimal loss of polymer molecular weight duringrecovery; and (iii) reduced residual solvent. There are no particularrestrictions on the weight average molecular weight of the P4HB polymer.However, in a preferred embodiment, the weight average molecular weightof the P4HB polymer ranges from 20 kDa to 1,200 kDa, more preferablyfrom 50 kDa to 800 kDa, and even more preferably from 200 kDa to 600kDa.

The P4HB polymer is extracted after washing the P4HB biomass with asuitable solvent to remove impurities, for example, lipid and heavymetal impurities.

The structure for P4HB is shown above. The compositions disclosed hereininclude polymers in which known isotopes of hydrogen, carbon and/oroxygen are enriched. Hydrogen has three naturally occurring isotopes,which include ¹H (protium), ²H (deuterium) and ³H (tritium), the mostcommon of which is the ¹H isotope. The isotopic content of the polymercan be enriched for example, so that the polymer contains a higher thannatural ratio of a specific polymer. The carbon and oxygen content ofthe homopolymer or copolymer can be also be enriched to contain higherthan natural ratios of isotopes of carbon, and oxygen, including, butnot limited to ¹³C, ¹⁴C, ¹⁷O or ¹⁸O. Other isotopes of carbon, hydrogenand oxygen are known to one of ordinary skill in the art.

A preferred hydrogen isotope enriched in P4HB is deuterium, deuteratedP4HB The percent deuteration can be up to at least 1% and up to 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85% orgreater.

A. P4HB Biomass

There is no particular restriction on the microorganism that can be usedprovided it is a microorganism that is capable of producing and storingP4HB in its cells. In a preferred embodiment, the P4HB biomass isconcentrated by centrifugation prior to purification, and refrigeratedor frozen. It is not necessary to completely dry the P4HB biomass,however, in a particularly preferred embodiment, the P4HB biomass isdried to a low moisture content of less than 5% residual water, or morepreferably less than 2% residual water. Suitable methods for drying thebiomass include, but are not limited to, spray drying, vacuum drying,lyophilization, or spray granulation.

Examples of suitable microorganisms that can be used as sources of P4HBbiomass, including mutated microorganisms and microorganisms geneticallymodified to produce P4HB, include microorganisms belonging to the genusEscherichia, Aeromonas, Alcaligenes, Azotobacter, Bacillus, Clostridium,Halobacterium, Nocardia, Nocardia, Actinomyces, Aguaspirillum,Paracoccus, Rhodospirillum, Pseudomonas, Ralstonia, Zoogloea Candida,Saccharomyces, and Yarrowia. Particularly preferred microorganisms forthe production of P4HB biomass include E. coli strain MBX1177, aderivative of strain DH5a selected for its ability to grow with4-hydroxybutyric acid as the sole carbon source, transformed with pFS30,a plasmid containing the genes encoding PHA synthase from Ralstoniaeutropha, 4-hydroxybutyryl-CoA transferase from Clostridium kluyveri,and β-lactamase, as disclosed by WO 99/32536 to Martin and WO 00/56376to Williams. Other microorganisms that may be used to produce P4HBbiomass include the mutant strain SK2813 derived from A. eutrophusJMP222 as disclosed by German Patent No. DE 3937649A1 to Steinbüchel etal., and the mutant strain of E. coli JM109 deficient in nativesuccinate semialdehyde dehydrogenase genes and harboring genes forsuccinate degradation from Clostridium kluyveri and PHB synthase fromRalstonia eutropha, together with genes for expression of four PHAbinding proteins, as disclosed by Zhou et al. Hyperproduction ofpoly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli,Microb. Cell Fact. 11:54 (2012).

Suitable P4HB producing microorganisms may be cultured by methods knownin the art, and reported as described above (for example by WO 99/32536to Martin, WO 00/56376 to Williams, EP 2534141 A1 to Van Walsem, WO2013/023140 to Van Walsem, and references therein), without anyparticular restrictions. Preferably, the microorganism and the culturingconditions are selected to yield high P4HB contents. In a particularlypreferred embodiment, the microorganisms contain at least 50 wt % P4HBmeasured as a percentage of dry cell weight.

4-hydroxybutyrate polymers enriched for specific isotopes of hydrogen,oxygen or carbon can be made using the same fermentation method used tomake PHA4400, however, using substrates that include the isotope ofchoice. For example, deuterated 1,4-butanediol [HO(CD₂)₄OH or1,4-butanediol1,1,2,2,3,3,4,4-d₈] can be used as a feed instead of1,4-butanediol. During the fermentation process, the deuterated1,4-butanediol is converted into deuterated 4HB (i.e. [²H₆]-4HB) andpolymerized into D-P4HB. The labeled polymer can be isolated andpurified by the same process used for PHA4400.

B. Washing Solutions Solvents

The P4HB biomass is preferably suspended in ethanol, and washed atambient temperature for one hour. The ratio of ethanol to P4HB biomassis preferably about 4 Kg of ethanol per Kg of P4HB biomass. The optimumamount of ethanol required to wash the biomass will be dependent uponthe P4HB biomass, the feedstock used to prepare the biomass, washingtime and temperature, moisture content of the biomass, and the amount oflipid and other impurities to be removed in the washing step. Aqueoussolutions of ethanol may also be used to wash the biomass althoughwashing with absolute ethanol is the preferred method. Alternatively,95% ethanol (190 proof ethanol) or other aqueous concentrations ofethanol may be used.

III. Methods of Extracting Higher Purity P4HB

Methods have been developed that allow P4HB to be recovered from P4HBbiomass with the following benefits: (i) higher purity; (ii) a goodyield of polymer; (iii) minimal loss of polymer molecular weight duringrecovery; (iv) fewer recovery steps; (v) reduced solvent usage duringextraction; (vi) easier drying of the polymer; (vii) lower cost; and(viii) faster overall process.

A. Washing of P4HB Biomass

It has been discovered that washing the P4HB biomass with ethanol priorto extraction of the P4HB polymer results in the removal of lipids andother impurities that can otherwise contaminate the extracted P4HBpolymer. Since ethanol is a poor solvent (i.e. non-solvent) for P4HB,but a good solvent for lipids, washing removes lipids but does notdissolve P4HB. It has also been discovered, that the P4HB biomass can bewashed with ethanol without causing any transesterification of the P4HBpolymer, and therefore the washing step may be done without anysignificant loss of polymer molecular weight. Furthermore, it has beendiscovered that washing the P4HB polymer with ethanol prior toextraction removes impurities that can cause a discoloration, oryellowing, of the purified product. Together, these improvements allowP4HB to be purified without multiple precipitation steps that arecommonly reported for the extraction of PHA polymers.

A major advantage of using ethanol to wash the P4HB biomass is itsclassification as a Class 3 solvent (International Conference onHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH) guidance for industry Q3CImpurities: Residual Solvents, 1997). Class 3 solvents are thosesolvents that are considered to have low toxic potential to man, andhave no set health based exposure limit.

In a preferred embodiment, the P4HB biomass is suspended in ethanol, andwashed at ambient temperature for one hour. The ratio of ethanol to P4HBbiomass is preferably about 4 Kg of ethanol per Kg of P4HB biomass. Theoptimum amount of ethanol required to wash the biomass will be dependentupon the P4HB biomass, the feedstock used to prepare the biomass,washing time and temperature, moisture content of the biomass, and theamount of lipid and other impurities to be removed in the washing step.Aqueous solutions of ethanol may also be used to wash the biomassalthough washing with absolute ethanol is the preferred method.Alternatively, 95% ethanol (190 proof ethanol) or other aqueousconcentrations of ethanol may be used.

In a particularly preferred embodiment, P4HB biomass derived fromrecombinant E. coli K12 is washed with ethanol to remove impurities. Theuse of ethanol to extract impurities from the E. coli K12 P4HB biomasshas been found to be very efficient. During the washing step, theethanol will typically become discolored with a yellow appearance asimpurities are extracted into the ethanol. These impurities have beenidentified as mostly saturated and unsaturated fatty acids by GCanalysis, with C16:0 and C18:1 fatty acids and oleate most prevalent. Atthe end of the washing step, the concentrated ethanol extract (which hasthe appearance of a black tar) has a high nitrogen content that willtypically be around 0.65 wt %. As such, the ethanol wash has been shownto remove nitrogen containing contaminants as well as lipids and coloredcontaminants.

After washing with ethanol, the biomass may be separated from theethanol wash by a method of solid-liquid separation, and collected byany suitable means. In a preferred embodiment, the P4HB biomass iscollected by filtration or centrifugation. If desired, additionalwashing of the collected P4HB biomass may be performed, or the P4HBbiomass may be rinsed with ethanol or aqueous ethanol during collection.Although it is not necessary to completely dry the P4HB biomass afterremoving the ethanol wash, in a preferred embodiment, the ethanol washedP4HB biomass is air-dried. In a particularly preferred embodiment, theP4HB biomass is dried to a residual ethanol concentration of between 1and 30 wt % ethanol. It has been discovered that the presence of higherquantities of residual ethanol in the washed P4HB biomass does notadversely impact the polymer recovery yield or the weight averagemolecular weight of the product.

In comparison to aqueous based extraction procedures, it has beendiscovered that washing the biomass with ethanol yields a P4HB biomassthat is easier to dry. When a dry P4HB biomass is required, washing withethanol not only removes impurities, it also displaces water from thebiomass, which significantly facilitates drying. As a result washingwith ethanol can save on the costs of drying, and speed up and simplifythe recovery process.

B. Extraction of P4HB Biomass

After the P4HB biomass has been washed with ethanol, the P4HB polymercan be extracted with a solvent. Ideally, the P4HB polymer has a highsolubility in solvents used to extract the polymer. Preferred solventsfor extracting the P4HB polymer from the ethanol washed biomass includemethylene chloride, chloroform, dichloroethane, tetrachloroethane,trichloroethane, dibromomethane, bromoform, tetrahydrofuran, acetone,dimethylformamide, and 1,4-dioxane. The ratio of solvent to biomassdepends upon the polymer content of the biomass, and on the nature ofthe solvent. If too little solvent is used the viscosity of theextracted polymer solution can become too high making further processingof the polymer solution difficult. In a particularly preferredembodiment, the amount of solvent needed is set so that a polymersolvent solution containing 2-5 wt % P4HB polymer is formed by the endof the extraction.

It has been discovered that the amount of solvent required to extractthe P4HB polymer with a good yield is significantly reduced when ethanolis used to wash the biomass prior to extraction of the polymer. Inaddition to saving the cost of additional solvent, the reducedextraction volume also decreases the amount of non-solvent for theP41113 polymer that is subsequently needed to precipitate the polymerfrom the solvent solution. Although it is preferred to precipitate thepolymer from solution to provide a polymer with the highest possiblepurity, reduced costs of solvent evaporation are realized if the polymeris simply concentrated after extraction and not precipitated.

C. Precipitation of P4HB Polymer and Drying

The P4HB polymer may be collected from solvent solutions of P4HB, whichhave been extracted from P4HB ethanol washed biomass, by precipitatingthe polymer with a non-solvent. This is preferable to crystallizing thepolymer from a solvent solution, which can require very large amounts ofsolvent in order to yield highly pure product and can consequently bevery expensive.

The non-solvent used to precipitate the P4HB polymer from a solventsolution of P4HB is preferably an alcohol or aqueous alcohol that is apoor solvent for the P4HB polymer. Water, ethanol, aqueous ethanol, andmethanol may be used to precipitate the P4HB, however, methanol is not apreferred non-solvent because of the potential toxicity of residues ofmethanol in the purified product. In a particularly preferredembodiment, the same type of solvent (or an aqueous solution of thesolvent) that is used to wash the P4HB biomass is also used as thenon-solvent to precipitate the P4HB polymer from the P4HB polymersolution. This is particularly desirable since it limits the number ofsolvents being used in the extraction process, and therefore limits thenumber of solvent residues that need to be removed and assayed in thefinal purified product. In a particularly preferred embodiment, ethanolis used to wash the P4HB biomass, and either ethanol or aqueous ethanolis used as a non-solvent to precipitate the polymer from a P4HB solventsolution. In an even more preferred embodiment, the P4HB biomass iswashed with ethanol, and the polymer is precipitated from a solventsolution of P4HB with aqueous solutions of ethanol containing 30-80% byweight of ethanol. The P4HB may be washed after precipitation withethanol or an aqueous solution of ethanol.

The ratio of non-solvent to the P4HB solvent solution that is requiredto precipitate the P4HB polymer will depend on the non-solvent, thesolvent for the P4HB, the temperature, the molecular weight of the P4HB,and the desired recovery yield. In a typical procedure, the ratio ofP4HB non-solvent to P4HB solvent ranges from 1:2 to 4:1, and is morepreferably closer to 1:1.

There are no particular limitations on the temperature that should beused to precipitate the P4HB polymer from the solvent solution, however,the temperature should be lower than the boiling point of the solventsolution and higher than its freezing temperature. In a preferredembodiment, the temperature of the precipitation step should be lessthan 50° C. and greater than 0° C., and more preferably at a temperatureof less than 25° C.

The precipitated P4HB polymer may be collected by any suitable means forseparating solids and liquids including the use of filtration andcentrifugation. The collected P4HB polymer may be further washed with anon-solvent for P4HB. In a preferred embodiment, the collected P4HBpolymer may be washed with ethanol or aqueous ethanol. Further washingwith ethanol may also be used to displace water from the collectedpolymer in order to make it easier to dry the P4HB polymer.

A major advantage of the method disclosed herein is that a highly pureP4HB polymer can be obtained with a single precipitation step.Additional precipitation steps may be performed by re-dissolving theP4HB polymer in a solvent, and repeating the precipitation procedure.However, in the preferred embodiment the P4HB polymer is purified withjust a single precipitation step, which eliminates the requirement touse large quantities of solvent for the P4HB purification.

After collecting the precipitated polymer, the P4HB may be desolventizedand dried by any suitable means. Suitable methods to remove residualsolvent and dry the polymer include air-drying and drying under vacuum.Desiccants may also be used to dry the polymer, and elevatedtemperatures can be used to shorten the time required to remove residualsolvent and dry the polymer.

D. Comparison of Purification Methods

In the procedures previously disclosed by WO 99/32536 to Martin and WO00/56376 to Williams, a five step process was used to purify P4HB. Thesteps include: (a) a step to fluidize and lyophilize the P4HB biomass,(b) a step to extract the P4HB polymer with tetrahydrofuran (THF) at 60°C., and filter insoluble matter, (c) a step to precipitate the P4HBpolymer into water, (d) a step to redissolve the P4HB polymer in solventand filter it again to remove insoluble matter, and (e) a step toprecipitate the polymer, wash the polymer, and lyophilize the polymer.This process yields a P4HB polymer with the following specification: (i)carbon content of 55.63%; (ii) hydrogen content of 7.41%; and nitrogencontent of 41 ppm. The lipid content of the P4HB polymer is notdisclosed in WO 99/32536 to Martin and WO 00/56376 to Williams, however,for comparison purposes it was determined as described in Example 5 andfound to be approximately 900 ppm palmitate fatty acid (see Example 6).

In contrast, the new methods developed and disclosed herein, allow theP4HB polymer to be purified in essentially three steps: (a) a step towash the P4HB containing biomass with ethanol, (b) a step to solventextract the P4HB biomass and filter insoluble matter, and (c) a step toprecipitate the P4HB polymer from the solvent with a non-solvent systemof aqueous alcohol, wash the P4HB polymer with ethanol or aqueousethanol, and desolventize and dry the P4HB polymer. Not only does thenew process have significantly fewer steps, it also yields P4HB polymerof higher purity. The Table below shows a side-by-side comparison of theP4HB extraction steps disclosed in WO 99/32536 and the extraction methoddescribed in this application

WO 99/32536 Disclosed method fluidize and lyophilize Wash Biomass withethanol Extract PHA with THF Solvent extract P4HB and filter insolublematter Precipitate P4HB into water — Redissolve P4HB in THF and — removeimpurities Precipitate P4HB into solvent, Precipitate P4HB from solventwash and lyophilize polymer into non-solvent, wash, desolventize and dry

In a preferred embodiment, the purity of the P4HB polymer purifiedaccording to the methods described in sections IIA, IIB and IIC meetsthe following specification: (i) carbon content of 55.81%±0.5%; (ii)hydrogen content of 7.02%±0.3%; (iii) lipid content of <100 ppm(measured as palmitate); (iv) residual solvent content <5 ppm; (v)4-hydroxybutyrate content of 99.7%±2% by weight; (vi) residue onignition of <0.2%; (vii) a heavy metal content of <20 ppm; and a sulfurcontent of <50 ppm. As shown in Example 2, the new process also yields aP4HB polymer with a nitrogen content of less than 40 ppm.

IV. Methods of Manufacturing Implants with High Purity P4HB

Implants made using high purity P4HB polymer have substantially improvedproperties for many medical applications. In particular, these implantshave low levels of organic impurities, inorganic impurities, andresidual solvents that can react with the body upon implantation. Thelow levels of these impurities will reduce or minimize undesirablereactions such as inflammation, cytotoxicity, irritation, pyrogenicity,subchronic and chronic toxicity. Devices made from or including highpurity P4HB may be prepared with endotoxin contents of less than 20endotoxin units per device.

Implants made from or including high purity P4HB polymer, and blendscontaining P4HB, may be used for soft and hard tissue repair,regeneration, and replacement. These implants may be used in medicaldevices, including but not limited to: suture, barbed suture, braidedsuture, monofilament suture, hybrid suture of monofilament andmultifilament fibers, braids, ligatures, knitted or woven meshes,knitted tubes, catheters, monofilament meshes, multifilament meshes,patches, wound healing device, bandage, wound dressing, burn dressing,ulcer dressing, skin substitute, hemostat, tracheal reconstructiondevice, organ salvage device, dural substitute, dural patch, nerveguide, nerve regeneration or repair device, hernia repair device, herniamesh, hernia plug, device for temporary wound or tissue support, tissueengineering scaffold, guided tissue repair/regeneration device,anti-adhesion membrane, adhesion barrier, tissue separation membrane,retention membrane, sling, device for pelvic floor reconstruction,urethral suspension device, device for treatment of urinaryincontinence, device for treatment of vesicoureteral reflux, bladderrepair device, sphincter muscle repair device, injectable particles,injectable microspheres, bulking or filling device, bone marrowscaffold, clip, clamp, screw, pin, nail, medullary cavity nail, boneplate, interference screw, tack, fastener, rivet, staple, fixationdevice for an implant, bone graft substitute, bone void filler, sutureanchor, bone anchor, ligament repair device, ligament augmentationdevice, ligament graft, anterior cruciate ligament repair device, tendonrepair device, tendon graft, tendon augmentation device, rotator cuffrepair device, meniscus repair device, meniscus regeneration device,articular cartilage repair device, osteochondral repair device, spinalfusion device, device for treatment of osteoarthritis, viscosupplement,stent, including coronary, cardiovascular, peripheral, ureteric,urethral, urology, gastroenterology, nasal, ocular, or neurology stentsand stent coatings, stent graft, cardiovascular patch, catheter balloon,vascular closure device, intracardiac septal defect repair device,including, but not limited to, atrial septal defect repair devices andPFO (patent foramen ovale) closure devices, left atrial appendage (LAA)closure device, pericardial patch, vein valve, heart valve, vasculargraft, myocardial regeneration device, periodontal mesh, guided tissueregeneration membrane for periodontal tissue, ocular cell implant,imaging device, cochlear implant, embolization device, anastomosisdevice, cell seeded device, cell encapsulation device, controlledrelease device, drug delivery device, plastic surgery device, breastlift device, mastopexy device, breast reconstruction device, breastaugmentation device (including devices for use with breast implants),breast reduction device (including devices for removal, reshaping andreorienting breast tissue), devices for breast reconstruction followingmastectomy with or without breast implants, facial reconstructivedevice, forehead lift device, brow lift device, eyelid lift device, facelift device, rhytidectomy device, thread lift device (to lift andsupport sagging areas of the face, brow and neck), rhinoplasty device,device for malar augmentation, otoplasty device, neck lift device,mentoplasty device, cosmetic repair device, and device for facial scarrevision. The devices may include a therapeutic, prophylactic, ordiagnostic agent.

These include, for example, compounds for the treatment, prevention,diagnosis, cure, or mitigation of one or more symptoms of a disease ordisorder, substances that affect the structure or function of the body,or pro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment. Theseinclude biologically, physiologically, or pharmacologically activesubstances that act locally or systemically in the human or animal body.Examples can include, but are not limited to, small-molecule drugs,proteins, peptides, sugars and polysaccharides, nucleic acids, lipids,and combinations thereof, which may function as anti-inflammatoryagents, immunomodulatory agents, molecules that affect cell migration,molecules that affect cell division, molecules that affect cellproliferation and differentiation, molecules that stimulate phenotypicmodification of cells, molecules that affect angiogenesis, moleculesthat affect vascularization, molecules that affect extracellular matrixdisposition, signaling ligands, antibodies, growth factors, integrins,antibiotics, steroids, hydroxyapatite, silver particles, vitamins, andnon-steroidal anti-inflammatory drugs. Materials may also includeextracellular matrix materials like fibronectin, laminin, vitronectin,chitosan and derivatives thereof, alginate and derivatives thereof,collagen, and hyaluronic acid and derivatives thereof. Nucleic acidsinclude antisense molecules, aptamers, siRNA, nucleic acids, andcombinations thereof.

The present invention will be further understood by referenced to thefollowing non-limiting examples.

Example 1: Washing of P4HB Biomass with Ethanol

A biomass containing P4HB (M_(w) of 468 kDa, by Gel PermeationChromatography (GPC) relative to standards of polystyrene), preparedaccording to Example 1 of WO 99/32536 to Martin, was suspended inethanol at room temperature. After one hour, the P4HB biomass wasremoved by filtration, and the ethanol wash concentrated to yield ablack tar. Analysis of the tar by NMR demonstrated that the ethanolextract of the P4HB biomass was composed almost entirely of saturatedand unsaturated lipids.

The nitrogen content of the tar was also determined to be 0.65 wt %.

Example 2: Purification of Ethanol Washed P4HB Biomass

The ethanol washed P4HB biomass derived from Example 1 was centrifugedin a basket centrifuge to remove the bulk of the ethanol washingsolution. The P4HB polymer was extracted into an organic solvent,precipitated into aqueous ethanol (30%), and collected for analysis. Thenitrogen content of the P4HB polymer extracted from the ethanol washedP4HB biomass was found to be 37 ppm as determined by the Kjeldahl method(Bradstreet, Anal. Chem., 26(1):185-187 (1954). The carbon and hydrogenmass fractions of the purified P4HB polymer were determined by elementalcombustion analysis using a LECO CHN 2000 instrument (following themanufacturer's instructions), and were found to be 55.89% and 7.05%,respectively. These values are close to the theoretical values forpoly-4-hydroxybutyrate: carbon 55.81%, and hydrogen 7.02%. Nosignificant loss of molecular weight of the P4HB polymer was observed.The weight average molecular weight of the purified P4HB polymer wasdetermined by GPC relative to polystyrene, and was found to be 449 kDa(versus 468 kDa prior to purification) indicating that the ethanolwashing step did not cause molecular weight loss by transesterificationof P4HB with ethanol. The purity of the P4HB polymer was determined byGC (gas Chromatography) analysis (as described in Example 3), and wasfound to be 99.5%. Proton NMR analysis of the purified P4HB polymerdemonstrated that the polymer was of high purity with little evidence ofcontaminating lipids at 1.2 ppm in the NMR spectrum. The endotoxincontent of the purified P4HB polymer was 0.22 endotoxin units (EU)/gwhich is low enough to allow the manufacture of implants using P4HB withan endotoxin content of less than 20 endotoxin units per device.

Example 3: Analysis of P4HB Purity by GC

The purity of a P4HB polymer in an unknown sample may be measured by gaschromatography after derivatization of the polymer using a butanolysisreaction to form volatile esters. The butanolysis reaction is anacid-catalyzed transesterification reaction with 1-butanol that convertsthe P4HB polymer into two major derivatives, butyl-4-hydroxybutyrate andbutyl-4-chlorobutyrate. The latter yields a sharp peak in the GCchromatograph that can easily be integrated, and its peak isproportional to the amount of P4HB in the sample.

The reagent for the butanolysis reaction is prepared by mixing equalparts (v/v) of 1-butanol and 4M hydrochloric acid (HCl) in 1,4-dioxanoneto yield a solution of 2M HCl in butanol/dioxane. An internal standard,such as diphenylmethane, may be added to the solution at a concentrationof 2.0 mg/ml to normalize injection volumes.

The butanolysis reaction is performed by adding 3 mL of the butanolysisreagent (prepared as described above) to a known mass of a P4HB sample(approximately 25 mg) in a vial. The vial is sealed, and heated at90-92° C. for 16-20 hours. (A standard curve may be generated bybutanolysis of known quantities of high purity gamma-butyrolactone(GBL)) After heating, the vials are allowed to cool, 3 mL of water areadded, and the contents of the vial are thoroughly mixed, and thenallowed to separate. The 4HB content in the sample may then bedetermined by GC analysis of the separated organic layer in the vial,using the GBL standard samples to create a standard curve.

The GC analysis is performed by injecting 1 μL of the organic phasecontaining the volatile butyl esters into a suitable gas chromatograph.One suitable GC set up comprises an Agilent 6890 GC (AgilentTechnologies, CA, USA) equipped with an autosampler, a flame ionizationdetector, and a SPB-35 capillary column from Supelco, Inc. (PA, USA) (30m×0.25 mm×0.25 microns) with helium used as a carrier gas at 2 ml/min.The inlet temperature is set at 225° C., and the split ratio is 50:1.The oven temperature program is set at 80° C. for 2 min, increasing 10°C. per minute to 280° C., and holding at 280° C. for 2 minutes. At thedetector, the temperature is set at 290° C., the hydrogen flow rate is40 ml/min, the helium makeup gas is set at 45 ml/min and the detectorair flow rate is set at 450 ml/min.

The mass of 4HB in the P4HB polymer is determined from integration ofthe sharp peak of butyl-4-chlorobutyrate in the GC chromatograph. Themass of 4HB in the sample can be determined from the GBL standard curve(plotted as mass vs. the integral area of the butyl-4-chlorobutyratepeak. The purity of the sample is determined as the mass percentage of4HB relative to the mass of polymer times 100%. The purity of P4HBpurified by methods described herein is 99.7+/−2 wt %.

Example 4: Additional Example of Purification of P4HB from P4HB Biomass

A P4HB biomass prepared according to Example 1 of WO 99/32536 to Martinet al. can be pre-washed with ethanol to remove colored contaminants andfatty acids prior to solvent extraction of the polymer using thefollowing procedure.

The dried P4HB biomass is suspended in absolute ethanol at a ratio of 1Kg of biomass to 4 Kg of ethanol, and stirred aggressively for one hourat room temperature. The resulting biomass slurry is transferred to abasket centrifuge, and the slurry is centrifuged to remove the washsolvent. During centrifugation, an additional wash with ethanol isperformed to further displace the wash solvent. Centrifugation isperformed to reduce the residual solvent content of the biomass to lessthan 15% by weight. After washing, the biomass is removed from thecentrifuge, analyzed for residual volatiles and collected for polymerextraction.

The collected washed biomass is transferred to an extraction vessel andextracted into a suitable solvent at an elevated temperature for 4hours. Suitable extraction solvents include polar organic solvents suchas chloroform, dichloromethane, dimethylformamide, tetrahydrofuran,acetone, dioxane, and mixtures of these. After extraction is completed,the extract is filtered to remove cell debris and insolubles, and thepolymer is precipitated by pumping the filtrate into a non-solvent forP4HB. A solution of ethanol and water (between 30-80% ethanol by wt) isused as the non-solvent. When the filtrate is pumped into this aqueousethanol solution, the P4HB precipitates as a solid, and is collected andfurther washed with ethanol. After washing, the collected P4HB polymeris transferred to a vacuum drying oven and dried at 45° C. under vacuum.

Example 5: Analysis of Fatty Acid (Lipid) Content of P4HB by GC

The analysis for fatty acids in a sample of P4HB is carried out in asimilar manner to the GC butanolysis purity analysis per Example 3,except that a fatty acid is used as a quantitative standard, rather thanGBL. The fatty acid palmitate, or its methyl ester, is a suitablestandard. The butanolysis reaction converts fatty acids, or fatty acidesters, into the corresponding fatty acid butyl esters. These volatileesters are analyzed by injection onto a GC as in Example 3. Palmiticacid, the most prevalent lipid in animals, plants and microorganisms, isused as a representative lipid or fatty acid to evaluate the purity andlipid content of a sample, even though other fatty acids or lipids mayalso be present in the sample. A standard curve for peak area of butylpalmitate vs. its mass is generated and used to determine the fatty acidcontent of the P4HB sample. Fatty acid concentration is reported as ppmpalmitate fatty acid.

Example 6: Determination of the Lipid Level in the P4HB Polymer PurifiedAccording to Example 1 of WO 99/32536 to Martin

Seven polymer samples of P4HB purified according to Example 1 of WO99/32536 to Martin were analyzed in triplicate for lipid content as inExample 5. The amount of lipid in the samples was found to range from304 to 2,207 ppm and the average was found to be 873 ppm, with astandard deviation of 629 ppm.

In comparison, the residual lipid content found in samples of P4HBpurified per the method of Example 2 was found to be less than 100 ppm.

Example 7: Determination of Heavy Metal Content, Residue on Ignition,Residual Solvent, and Sulfur Content in High Purity P4HB Polymer

P4HB polymer purified according to the method described herein wasanalyzed for heavy metal content, residue on ignition, the presence ofresidual solvent, and sulfur content. The heavy metal content of thepolymer was determined by USP <231> (a colorimetric procedure based onthe precipitation of insoluble metal sulfides) and was found to be <20ppm. The residue on ignition of the purified polymer was determined byUSP <281> and was found to be <0.1%. The amount of residual solvent inthe polymer was measured by Headspace GC-MS using an HP 5890 II GCequipped with a 5972 MS Detector and a ZB-5 Capillary Column (60 m×0.32mm ID×1 μm FT). Samples of polymer were heated in capped vials at 130°C. for one hour before analysis. The residual solvent content of theP4HB polymer was found to be <5 ppm. The sulfur content of the purifiedP4HB polymer was determined by inductively coupled plasma opticalemission spectroscopy, and found to be <50 ppm.

Example 8: Preparation of Deuterated P4HB (D-P4HB) Implants andImplantation Procedure

Experiments were conducted to quantify the release of 4-hydroxybutyricacid (4HB) during the degradation and absorption of a PHA4400(TephaFLEX) implant. During this study, the blood concentration ofdeuterium labeled 4HB (D-4HB) was monitored in rabbits that had beenimplanted with deuterium labeled PHA4400 (D-PHA4400). The bloodconcentration of the native (i.e. unlabeled) 4HB was also monitored todetermine if the D-4HB released from a D-PHA4400 implant affected thenative levels of 4HB. Additionally, the rabbits' blood was analyzed fortraditional clinical blood parameters.

A PHA4400 implant will degrade in the body primarily by hydrolysis toproduce 4HB. This monomer is a normal constituent of the mammalian bloodserum and is found within a variety of tissues, including brain, heart,kidney, liver, lung, muscle and brown fat (Nelson, et al., J.Neurochem., 37:1345-1348 (1981)). The concentration of 4HB in thesetissues ranges from 2.3 to 37.4 nmoles/g of tissue (or 0.24 to 3.8milligrams per Kg of tissue, based on a formula weight of 104 g/mol).The body quickly metabolizes 4HB, and its half-life is approximately 27minutes (Nelson, et al., J. Neurochem., 37:1345-1348 (1981); Sendelbeck,et al., Drug Metab. dispos. 13:291 (1985)). 4HB is eliminated from thebody primarily by metabolism (via the Krebs Cycle) and secondarily bybeta-oxidation ultimately to carbon dioxide and water.

D-P4HB was made using the same fermentation method disclosed herein.However, deuterated 1,4-butanediol [HO(CD₂)₄OH or1,4-butanediol,1,1,2,2,3,3,4,4-d₈] was used as a feed instead of1,4-butanediol. During the fermentation process, the deuterated1,4-butanediol was converted into deuterated 4HB (i.e. [²H₆]-4HB) andpolymerized into D-P4HB. The labeled polymer was isolated and purifiedby the same process used for P4HB. NMR spectra for the D-P4HB (Lot No.DM21.87) showed that the ¹³C NMR spectrum of the labeled polymer is abit more complicated than the unlabeled polymer since each carbonattached to deuterium is split into a multiplet. The carbonyl (no boundD) appears as a singlet. The ¹H NMR spectrum of the labeled polymer doesnot show the deuterium, but rather shows residual protons at theirexpected chemical shifts.

Elemental analysis of the polymer showed the level of carbon to be52.08%, which is within the acceptable limits of the expected value of52.20% (±0.5%) for the repeating formula of C₄D₆O₂ calculated for heavyisotope. The weight percentage of deuterium was not calculated. Thepolymer was put through an additional round of purification to yield anew lot (Lot No. 225-030). The Mw of this new lot was determined to be525 K. GC analysis for lot no. 225-030 passed the purity assay (98.3%purity).

Implantation Method

Eight rabbits were used for the study. Six (6) disks of D-PHA4400material (M6) were implanted subcutaneously in the back of each of sixrabbits, so each animal was implanted with approximately 341 mg ofD-PHA4400. The other two rabbits were implanted with 6 disks of highdensity polyethylene (HDPE) as controls. To facilitate later recovery ofthe implants, the peri-incision areas were tattooed.

Following surgery, one rabbit in the study (rabbit 8) was found to havea hard mass on the abdomen a few days after implantation. Treatment withantibiotics was unsuccessful and the animal was euthanized at day 28.Due to its location in the abdominal muscle, this mass was most likelynot implant related, but is believed to have pre-dated the implants. Toreplace this animal, an additional animal (rabbit 9) was added to thestudy at 1 month. The recovery of the other animals (1-7, 9) in thestudy was uneventful.

The rabbits were maintained for a period of 1 year and blood was drawnfrom each rabbit monthly for analysis of [²H₆]-4HB and 4HBconcentrations in the serum. Blood samples were collected using fluorideanticoagulation, centrifuged and the serum samples is stored frozen at−20° C. for batch analysis at 3 and 6 months and 1 year. Clinical bloodchemistry parameters were determined at pre-implant, 3 months, 6 monthsand 1 year (Note: Additional clinical testing was done at 4 and 5months). At the conclusion of the study, the animals were sacrificed abdany residual implant material harvested for analysis of mass loss andMw. Tissue samples taken from the implantation sites of each animal wereprocessed for histological analysis.

Blood Analysis Methodology To analyze for the presence of 4HB in theblood, serum samples (0.5 ml) were spiked with 8.6 nmol of5-hydroxypentanoate (i.e. valerolactone) as an internal standard,deproteinized with sulfosalicylic acid, centrifuged and extracted withether (3 times). Pooled extracts were dried over sodium sulfate,concentrated and hydrolyzed with NaOH (1 ml, 10 mM). After evaporation,the residue was converted to the trimethyl silyl derivative. Capillarygas chromatography-mass spectrometry (GC-MS) analysis was performedusing a Hewlett-Packard MS-engine in the ammonia positive chemicalionization mode. Ions were monitored for unlabeled 4HB at m/z 249, [²H₆]4HB at m/z 255 and the valerolactone internal standard at m/z 263, asthe trimethyl silyl derivatives. A reference standard of [²H₆] 4HB wasprepared by hydrolysis of the D-PHA4400. The conditions of the assaywere optimized by preparation of standard curves using spare samples ofrabbit plasma. The temperature profile of the GC column was optimized sothat the 3 compounds elute free of interferences from unknown compoundsand this was verified by the identity of the mass isotopomerdistributions of the 3 compounds in pure aqueous solutions and in plasmaextracts. The standard curve was linear in the range of 0 to 15 nmol andis shown in FIG. 1A.

Results

Serum samples were analyzed through 6 months of the study. The averagebasal serum concentration of 4HB (i.e. prior to implantation of theD-PHA4400 and control disks) was found to be 10.6±2.9 nmol/ml. Duringthe 6-month observation period, there was no significant difference inthe serum concentration of 4HB between the control and the D-PHA4400implanted animals. Over the 6-month observation period, the serumconcentration of 4HB decreased to approximately 4 nmol/ml in both thecontrol and the D-PHA4400 implanted animals, as shown in FIG. 1B andTable 1.

TABLE 1 Blood concentration of 4HB following implant of deuteriumlabeled PHA4400 or HDPE disks subcutaneously in a rabbit model. TimeD-PHA4400 Implant HDPE Implant Control Months 4HB Blood Conc. (nmol/ml)4HB Blood Conc. (nmol/ml) 0 10.60 ± 2.89  10.60 ± 2.89  1 11.65 ± 2.58 13.82 ± 2.83  2 10.88 ± 2.11  10.87 ± 3.34  3 8.35 ± 1.84 8.90 ± 0.41 45.30 ± 2.35 3.29 ± 2.43 5 4.22 ± 1.78 3.22 ± 0.76 6 4.79 ± 1.56 4.66 ±2.38

Post implantation surgery, the concentration of [²H₆]-4HB in the bloodserum from all but one of the test animals was below the detection limitof the assay (approximately 1 nmol/ml). In one animal (rabbit 6), theconcentration of [²H₆]-4HB in the blood serum was found to be 2.52 and1.88 nmol/ml at 1 month and 2 months post implantation, respectively.These concentrations represent 18-24% of the basal level of the naturalmetabolite GHB at time zero. This amount of [²H₆]-4HB is less than thestandard deviation observed in the basal level of GHB and does not causea significant change in the total blood serum concentration of GHB(labeled and unlabeled) since the basal concentration of GHB ranged from6.95 to 15.79 nmol/ml (10.8±2.89 nmol/ml). At 3 to 6 months postsurgery, none of the animals had detectable levels of [²H₆]-4HB in theirblood serum.

TABLE 2 Serum concentration of [²H₆]-4HB after subcutaneous implantationof D-PHA4400 or HDPE polymer disks in a rabbit model Rab- BasalConcentration of [²H₆]-4HB (nmol/ml) bit Implant (Pre- Month ID Materialimplant) 1 2 3 4 5 6 2 D-PHA4400 N.D. N.D. N.D. N.D. N.D. N.D. N.D. 3D-PHA4400 N.D. N.D. N.D. N.D. N.D. N.D. N.D. 4 D-PHA4400 N.D. N.D. N.D.N.D. N.D. N.D. N.D. 5 D-PHA4400 N.D. N.D. N.D. N.D. N.D. N.D. N.D. 6D-PHA4400 N.D. 2.52 1.88 N.D. N.D. N.D. N.D. 9 D-PHA4400 N.D. N.D. N.D.N.D. N.D. N.D. N.D. 1 HDPE N.D. N.D. N.D. N.D. N.D. N.D. N.D. 7 HDPEN.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.—Not detected. Note: Rabbit 8 waseuthanized at day 28. Rabbit 9 was implanted 1 month after rabbits 1-8.

Clinical Chemistry

In addition to the blood serum analysis for 4HB and D-4HB, the serumsamples were analyzed for standard clinical parameters (bicarbonate,BUN, Creat, Glucose, AST, ALT, alk phos, alb, TB, Db, and totalprotein). These values were unremarkable except for rabbit 6 whichshowed a reduced bicarbonate level (16 meq/l) at pre-implant and anelevated BUN (40 mg/dl) at 3 months post implant (Table 3). These valueswere attributed to a slight renal deficiency in this animal, and werejudged clinically irrelevant. Notably, this animal (rabbit 6) was theonly rabbit to show a measurable amount of D-4HB in its blood serumalthough the level of D-4HB measured was very low and near the detectionlimit.

TABLE 3 Clinical blood parameters of serum following implantation ofD-PHA4400 or HDPE (control) measured at time 0 and at 3 months. HCO3 BUNCreat Glu AST ALT alk phos alb TB Db Total protein Normal values 22-268-25 0.6-1.5 9-40 7-55 <200 3.1-4.3 Billirubin Billirubin 6-8 meq/lmg/dl mg/dl mg/dl U/L U/L U/L g/dl 0-1 mg/dl 0-0.4 mg/dl g/dl CONTROLSJuly 19 rabbit 1 24 17 0.9 99 22 37 44 1.6 <0.1 <0.1 5.2 July 19 rabbit7 23 11 0.8 126 9 28 158 1.5 0.1 0.1 4.8 AVERAGE 23.5 14 0.85 112.5 15.532.5 101 1.55 0.1 0.1 5 CONTROLS Oct 25 rabbit 1 22 22 1 108 22 51 201.6 0.1 <0.1 4.9 Oct 25 rabbit 7 25 21 1 118 9 37 53 1.9 0.2 0.1 5.5AVERAGE 23.5 21.5 1 113 15.5 44 36.5 1.75 0.15 0.1 5.2 poly-GHBimplanted rabbits July 20 rabbit 2 22 21 0.9 122 10 23 59 1.7 0.1 <0.15.1 Oct 25 rabbit 2 21 21 1 119 13 31 49 1.8 0.1 <0.1 5.3 July 20 rabbit3 19 17 1 122 20 34 301 1.9 <0.1 <0.1 5.7 Oct 25 rabbit 3 21 22 0.9 11319 27 113 1.8 0.1 <0.1 5.4 July 19 rabbit 4 20 17 0.7 103 20 34 97 1.6<0.1 <0.1 4.8 Oct 25 rabbit 4 21 17 0.8 100 10 32 51 1.6 <0.1 0.1 4.8July 19 rabbit 5 22 16 1 106 9 41 51 1.6 0.1 0.1 4.7 Oct 25 rabbit 5 2720 1 118 4 31 34 1.8 0.1 0.1 5.3 July 19 rabbit 6 14 17 1.2 108 20 17 691.5 0.2 0.1 5.1 Oct 25 rabbit 6 23 41 1.4 109 6 21 18 1.9 0.1 0.1 5.6rerun July 19* rerun rabbit 6 16 19 1.3 108 13 17 86 1.7 0.3 0.1 5.6rerun Oct 25* rerun rabbit 6 21 40 1.4 103 8 20 23 1.8 0.2 0.1 5.7 Aug19 rabbit 9 24 18 1 108 15 42 79 1.6 <0.1 0.1 5 Oct 25 rabbit 9 24 21 1119 6 28 56 1.7 0.1 0.1 5.2 Average July rabbit 2, 3, 5, 6, 9 20.2 17.71.0 111.5 15.7 31.8 109.3 1.7 0.1 0.1 5.1 Std dev July 3.2 1.6 0.1 7.64.7 9.1 87.0 0.1 0.0 0.0 0.3 Average October rabbit 2, 3, 5, 6, 9 22.823.7 1.0 113.0 9.7 28.3 53.5 1.8 0.1 0.1 5.3 Std dev October 2.2 7.9 0.26.9 5.1 3.7 29.5 0.1 0.0 0.0 0.2

Conclusion of Bioavailability Study

Blood serum analysis of rabbits implanted with D-PHA4400 showed nosignificant change in the blood concentration of native 4HB compared tothe control rabbits implanted with HDPE. Additionally, the blood serumconcentration of D-4HB remained below the detection limit (approx. 1nmol/ml) in all but one animal. One animal (rabbit 6) showed adetectable level of D-4HB in its blood serum at 1 and 2 months postimplantation, however, the concentration was below the detection limitat 3 to 6 months in this animal and otherwise in all other animals. Assuch, the release of 4HB from a PHA4400 implant does not affect thenative level of 4HB in the blood, nor does it accumulate in the blood.

The blood chemistry of the animals implanted with PHA4400 remainednormal during the 6-month observation period. One animal (rabbit 6)showed slightly reduced level of bicarbonated and a slight elevated BUNpre-implant and at 3 months, respectively. These values were not thoughtto be clinically relevant as the animal's clinical blood parametersreturned to normal at 4 months and the animal was otherwise healthy. Inconclusion, release of 4HB from a PHA4400 implant does not adverselyaffect the standard clinical blood parameters measured in this study.

Since 4HB is non-toxic, released slowly from an implant, and can bereadily metabolized, the amount of 4HB released from a PHA4400 implantduring its degradation in the body is expected to be safe and welltolerated by the body.

The amount of D-PHA4400 implanted into each rabbit in this study wasapproximately 341 mg. Since the average weight of a rabbit isapproximately 3 to 3.5 kg, the amount of D-PHA4400 implanted isapproximately 100 mg per kg of body weight. On a per mass basis, thiswould be similar to implanting upwards of 6 g of PHA4400 into a person,assuming the person weighed say 60 kg. Many medical devices made fromPHA4400 are expected to weigh less than 6 g, so the amount of PHA4400used in this rabbit study is representative of a fairly massive medicaldevice made of PHA4400.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A composition comprising a polyhydroxyalkanoate (PHA)polymer obtained by a process comprising: suspending a PHA polymerbiomass in ethanol for a period of time effective to extract lipids intothe ethanol, separating the PHA polymer biomass from the ethanol bysolid-liquid separation, collecting the ethanol-washed biomass, andextracting the PHA polymer into a solvent.
 2. The composition of claim 1wherein the polymer is precipitated with a non-solvent for the polymer,harvested, and dried.
 3. The composition of claim 1 wherein the weightaverage molecular weight of the polymer is from 50,000 to 1.2 millionDa.
 4. The composition of claim 1 sterilized with cold ethylene oxidegas, gamma-irradiation, or electron beam irradiation.
 5. The compositionof claim 4 used for soft or hard tissue repair, regeneration orreplacement.
 6. The composition of claim 1 wherein the composition isused as a coating on a medical device.
 7. The composition of claim 1wherein the composition is used to form a medical device.
 8. Thecomposition of claim 1 wherein the PHA polymer ispoly-4-hydroxybutyrate.
 9. The composition of claim 8 wherein thecomposition forms a fiber wherein the fiber has a tensile strength ofgreater than 126 MPa.
 10. The composition of claim 8 wherein thecomposition is used to form a yarn wherein the tenacity of the yarn isgreater than 0.5 grams per denier.
 11. The composition of claim 9 or 10formed into a suture or textile.
 12. The medical device of claim 7wherein the device is selected from the group: suture, barbed suture,braided suture, monofilament suture, hybrid suture of monofilament andmultifilament fibers, braids, ligatures, knitted or woven meshes,knitted tubes, catheters, monofilament meshes, multifilament meshes,patches, wound healing device, bandage, wound dressing, burn dressing,ulcer dressing, skin substitute, hemostat, tracheal reconstructiondevice, organ salvage device, dural substitute, dural patch, nerveguide, nerve regeneration or repair device, hernia repair device, herniamesh, hernia plug, device for temporary wound or tissue support, tissueengineering scaffold, guided tissue repair/regeneration device,anti-adhesion membrane, adhesion barrier, tissue separation membrane,retention membrane, sling, device for pelvic floor reconstruction,urethral suspension device, device for treatment of urinaryincontinence, device for treatment of vesicoureteral reflux, bladderrepair device, sphincter muscle repair device, injectable particles,injectable microspheres, bulking or filling device, bone marrowscaffold, clip, clamp, screw, pin, nail, medullary cavity nail, boneplate, interference screw, tack, fastener, rivet, staple, fixationdevice for an implant, bone graft substitute, bone void filler, sutureanchor, bone anchor, ligament repair device, ligament augmentationdevice, ligament graft, anterior cruciate ligament repair device, tendonrepair device, tendon graft, tendon augmentation device, rotator cuffrepair device, meniscus repair device, meniscus regeneration device,articular cartilage repair device, osteochondral repair device, spinalfusion device, device for treatment of osteoarthritis, viscosupplement,stent, including coronary, cardiovascular, peripheral, ureteric,urethral, urology, gastroenterology, nasal, ocular, or neurology stentsand stent coatings, stent graft, cardiovascular patch, catheter balloon,vascular closure device, intracardiac septal defect repair device,including but not limited to atrial septal defect repair devices and PFO(patent foramen ovale) closure devices, left atrial appendage (LAA)closure device, pericardial patch, vein valve, heart valve, vasculargraft, myocardial reneration device, periodontal mesh, guided tissueregeneration membrane for periodontal tissue, ocular cell implant,imaging device, cochlear implant, embolization device, anastomosisdevice, cell seeded device, cell encapsulation device, controlledrelease device, drug delivery device, plastic surgery device, breastlift device, mastopexy device, breast reconstruction device, breastaugmentation device (including devices for use with breast implants),breast reduction device (including devices for removal, reshaping andreorienting breast tissue), devices for breast reconstruction followingmastectomy with or without breast implants, facial reconstructivedevice, forehead lift device, brow lift device, eyelid lift device, facelift device, rhytidectomy device, thread lift device (to lift andsupport sagging areas of the face, brow and neck), rhinoplasty device,device for malar augmentation, otoplasty device, neck lift device,mentoplasty device, cosmetic repair device, and device for facial scarrevision.
 13. The medical device of claim 12 wherein the PHA polymer ispoly-4-hydroxybutyrate.
 14. The medical device of claim 13 comprising afiber wherein the fiber has a tensile strength of greater than 126 MPaor a yarn wherein the tenacity of the yarn is greater than 0.5 grams perdenier.
 15. A process for purifying PHA polymer from a biomasscomprising suspending a PHA polymer biomass in ethanol for a period oftime effective to extract lipids into the ethanol, separating the PHApolymer biomass from the ethanol by solid-liquid separation, collectingthe ethanol-washed biomass, and extracting the PHA polymer into asolvent.
 16. The process of claim 15, comprising suspending the biomassin ethanol for one hour at ambient temperature.
 17. The process of claim15 wherein the wt/wt ratio of P4HB biomass to ethanol is 1:1.
 18. Theprocess of claim 16 wherein the biomass is washed with at least 4 gramsof ethanol per gram of biomass.
 19. The process of claim 15, furthercomprising precipitating the polymer from the solvent with a non-solventfor the polymer, and drying the polymer.
 20. The process of claim 15wherein the solvent is selected from the group consisting of methylenechloride, chloroform, tetrahydrofuran, acetone, dioxane, ethyl acetate,dimethylene carbonate, dimethyl sulfoxide, dimethyl formamide, methylethyl ketone, butyl acetate, butyl propionate and diethyl carbonate, andwherein the non-solvent for the polymer is an alcohol, aqueous alcohol,or water.
 21. The process of claim 19, the ratio of P4HB non-solvent toP4HB solvent ranges from 1:2 to 4:1, and is more preferably 1:1.